Manilagrass with green leaves in winter and eragrostoideae plant produced therefrom

ABSTRACT

A series of independent technical systems including a combination of mutation methods such as cellular mutation, ultraviolet irradiation, X-ray irradiation or the like, formation and use of shoot primordium and selection of mutated cells are employed to produce genetic mutation in Manilagrass and obtain Manilagrass that retains its green leaves in winter while producing substantially no anthocyanins under normal cultivating conditions, Manilagrass that has a high stolon density, and dwarfed Manilagrass; the newly invented completely novel genotypes are used to obtain new Eragrostoideae plants.

TECHNICAL FIELD

The present invention relates to plants belonging to a novel Manilagrassvariety newly produced by adding a genetic variation to a single line(strain) of Zoysia matrella (common name: Manilagrass) withoutconventional crossbreeding, cell fusion, gene introduction or the like,which variety retains the characteristics of conventional Manilagrasswhile also exhibiting a completely new character. Specifically, itrelates to a novel Manilagrass that retains its green leaves under thesame seasonal conditions in which conventional Manilagrass loses itsgreen leaves. The present invention further relates to Manilagrass thatretains its green leaves in winter while producing substantially noanthocyanins under normal cultivating conditions, Manilagrass that has ahigh stolon density, dwarfed Manilagrass, and newly inventedEragrostoideae plant developed using genotypes absent in conventionalManilagrass lines but present in Manilagrass of the invention.

BACKGROUND ART

“Manilagrass” according to the present invention refers to Zoysiamatrella that exhibits creeping properties by vegetative growth, havinga blade width of 1.0-3.5 mm, and it does not include interspecies hybridlines obtained by artificial crossbreeding with other species such asZoysia japonica, Zoysia tenuifolia and Cynodon dactylon (see Fukuoka,H., “Turfgrass and Its varieties”, ed. by Asano, Y. and Aoki, K., Jun.15, 1998, Softscience Publications, pp.122-123).

Zoysia matrella (common name: Manilagrass) is widely used as a groundcover for a broad range of purposes, because of its many featuresincluding aesthetic appearance, ground spread, management ease, wearresistance, water stress resistance, growing power and creepingproperties.

However, Manilagrass has certain disadvantages including withered leafduring winter, strong preferential growth of main apical buds, impairedappearance due to anthocyanins, and the need for more frequent trimmingin summer due to intensified growth; for these reasons, it not onlyfails to satisfy market needs but is also limited in its uses.

Specifically, for uses that place importance on aesthetic appearance,for example, sports turfs, open areas such as parks, factory lawns andfacility exteriors, rooftop heat-insulating greenery, gardens and thelike, withered leaf during winter notably lowers the value ofManilagrass, and the concept of “evergreen Manilagrass” has been arequirement for numerous purposes and would meet a very strong marketdemand.

In order to compensate for withered leaf in winter of Manilagrass andBermudagrass (Cynodon dactylon), many sports turfs are managed with theoverseed method (a method in which Western grass, which is a cool seasonturfgrass, is sown over Manilagrass areas or Bermudagrass areas at thebeginning of autumn in order to maintain the green appearance in winter,and then the western grass is killed off in spring to allow resproutingof the Manilagrass or Bermudagrass), or the withered leaf coloringmethod (a method in which the withered leaves of Manilagrass are coloredwith a green pigment or dye). These methods, however, require expensiveinvestment each year leading to notable cost increase and, although theyare often employed at golf courses and other sports turfs they aredifficult to carry out in practice in most other fields, while theoverseeding method also results in yearly weakening of the Manilagrassitself.

Withered leaf in winter of Manilagrass is also a fatal drawback forrooftop heat-insulating greenery, used for its effectiveness towardenergy reduction and carbon dioxide gas fixation.

Withered leaf in winter of Manilagrass and Bermudagrass tends to resultin bare ground due to the wearing that occurs with trampling, etc., anda further drawback, in the case of Manilagrass, is its susceptibility towinter weeds and spring sprouting weeds, which tend to promote turfgrassdecay.

In addition to physical methods such as the overseed method and thewithered leaf coloring method, the following measures have also beenadopted to shorten the withered leaf in winter period of Eragrostoideaeturfgrass.

(a) Dispersion of iron chemicals: Iron absorption promotes chlorophyllsynthesis to increase the greenness of the plants.

(b) Dispersion of nitrogen fertilizers: This method is effective formaintaining greenness during the initial winter period but it alsodelays sprouting in spring, and because the withered leaf in winterperiod is not significantly shortened it is not a preferred method.

(c) Dispersion of 5-aminolevulinic acid (ALA): This has been reported tohave a chlorophyll-increasing effect (“5-Aminolevulinic Acid:Applications for Microbe Production and Lawngrass”, Hotta, Y., Tanaka,T., Watanabe, K., Takeuchi, Y., Konnai, M., “Lawngrass Research” meetingjournal, No.27, 1998, pp.138-139).

(d) Breeding methods: It has been attempted to crossbreed Zoysiamatrella and Zoysia japonica, for example, to solve the problem ofwithered leaf in winter. Further collection and selection of regionallines throughout the world have also been attempted in order to obtainvarieties with favorable genotypes, for example, by selection of lineswith minimal withered leaf in winter. (For example, Fukuoka, H.,“Turfgrass and Its Varieties”, ed. by Asano, Y. and Aoki, K.,Softscience Publications, Chap. 3, 3-2, pp.126-130). However,crossbreeding Zoysia matrella with other varieties tends to lessen thefeatures of Manilagrass, with the resulting varieties having wider bladewidths and inferior aesthetic appearance compared to Zoysia matrella,while the problem of withered leaf in winter is not satisfactorilysolved. On the other hand, methods involving the latter collection oflines mentioned above have to date failed to provide Manilagrass withsufficient greenness in winter.

DISCLOSURE OF THE INVENTION

The present invention relates to Manilagrass characterized by retainingits green leaves under a condition where the mean temperature of aperiod of ten days is 6° C. or below and the lowest temperature in thisperiod is −1° C. or below, but not less than −15° C., and by containingsubstantially no anthocyanins throughout the year.

The invention further relates to the aforementioned Manilagrass,characterized in that the length of the internode of a main stolonexcept the immature intemodes of the front part of the main stolon,which extends when attached to the soil surface under obstacle-freegrowth conditions, is about 0.9—about 0.6, where 1.0 is defined as thelength for conventional Tottori Z. matrella (Tottori Sod ProducersAssociation: the representative Manilagrass single line produced andmarketed at 558-1 Oaza-Tokuman, Tohaku-cho, Tohaku-gun; Tottori, Japan).

The invention still further relates to each aforementioned Manilagrass,characterized in that the ratio of the main stolon length to the totallateral stolon length, measuring the total length of lateral stolonsdeveloping from the main stolon based on a main stolon lengthcorresponding to at least 20 nodes from the tip of the main stolon ofthe turigrass of the invention, in stolons extending under obstacle-freegrowth conditions when attached to the soil surface, is at least 1.2times compared to conventional Tottori Z. matrella.

The invention still further relates to Eragrostoideae plant bred fromany of the aforementioned Manilagrass as the parental strain bycrossbreeding, mutation, cell fusion or gene introduction, that inheritsany of the aforementioned characteristics.

The novel Manilagrass of the invention has notably higher lateral stolonextendibility compared to the parental strain (Tottori Z. matrella) orJapanese lawngrass (Zoysia japonica), and thus has a higher stolondensity, forms a stratified mesh mat earlier and has much higher valuefor practical use.

Formation of a stratified mesh mat offers the following merits:

(1) Because growth (vegetative propagation) of Manilagrass by seed isdifficult in practice, productivity can be improved as the methodadopted is to strip a 1-2 cm upper layer, as harvest, each year from theproduction farm, and repopulate the grass from the remaining mat forharvest the following year;

(2) It is resistant to wearing;

(3) It can be rapidly reproduced even when the upper layer portion hasbeen cut away (for example, as golf course divots);

(4) It increases cushioning properties for sports turf;

(5) It improves drying resistance properties (water retention, waterabsorption, water storage in plant body);

(6) It greatly improves the spread essential for slopes, riverbeds,etc., and thus helps protect surface soil layers;

(7) It is dense and gives attractive lawn tops;

(8) It can reduce weeds by the formation of dense turfs.

In addition, conventional Manilagrass produces anthocyanins and colorsin the stems throughout the year and undergoes coloration, whileanthocyanins are produced resulting in coloration even in the few livingleaves remaining under daily mean temperatures of lower than about 10°C., such that it presents a purplish red, dark green-purple orblackish-purple color, thereby losing much of its attractive greenappearance. According to the present invention, however, theaforementioned problem is solved by providing the first variety ofManilagrass to contain substantially no anthocyanins.

In order to ensure high quality lawn tops for golf course fairways andthe like, they must be mowed about 2-3 times per week during the hightemperature growth season, and in some cases growth retardants aredispersed on purpose to reduce the mowing frequency. This leads to anotable increase in maintenance costs. The Manilagrass of the inventionalso provides an improvement in this respect.

It is an object of the present invention to provide a new variety ofManilagrass that retains its green leaves in winter while containingsubstantially no anthocyanins throughout the year, as well as dwarfedManilagrass, Manilagrass that has a high stolon density, andEragrostoideae plant incorporating genotypes of the Manilagrass newlydeveloped according to the invention.

For the purpose of the present invention, the term “winter” will referto the low temperature period in which conventional Manilagrass, whichis the starting material for the invention, experiences loss of greenleaves and undergoes coloration to brown or dried grass color undercommon practical outdoor cultivating conditions, and for example,Tottori Z. matrella at the research field of Kaisui Chemical IndustryCo., Ltd. experiences withered leaf in early January, becoming driedgrass colored across the entire covered region.

The mean measured temperature of a period of ten days for early January,1999 was 6.6° C., with a low temperature of −0.8° C. Table 1 shows theweather conditions from mid December, 1998 to early March, 1999 at theabove-mentioned research field.

TABLE 1 Weather conditions from Dec. 11, 1998 to Mar. 10, 1999 atresearch field of Kaisui Chemical Industry Co., Ltd. Mean Highest LowestTotal temperature temperature temperature sunlight Yr.Mon.Day (° C.) (°C.) (° C.) (MJ/m²) 1998 12 11 5.7 10.4 1.7 5.2 12 5.6 11.2 1.4 8.9 137.3 14.0 1.4 7.6 14 9.8 15.1 2.8 5.4 15 10.3 13.3 6.4 4.5 16 8.4 14.24.2 5.3 17 8.6 16.7 3.4 8.8 18 8.3 15.6 3.3 7.0 19 10.1 16.8 3.8 6.7 2010.4 14.4 4.6 9.0 Mid ten 8.5 16.8 1.4 68.4 days of the month 21 6.913.7 1.5 9.0 22 8.4 16.5 2.5 8.7 23 9.7 15.2 4.7 6.3 24 8.4 11.5 5.0 2.525 6.8 11.4 3.1 9.0 26 7.9 13.9 2.8 8.4 27 8.1 13.8 4.4 4.4 28 8.0 13.83.4 6.8 29 8.0 14.5 3.7 5.6 30 6.9 11.0 4.2 9.0 Late ten 7.9 16.5 1.569.7 days of the month 12 31 5.7 9.0 1.7 7.0 1999 1 1 4.4 10.1 0.2 8.2 25.3 9.5 0.0 1.9 3 6.0 12.4 0.9 5.3 4 6.3 12.9 1.2 7.3 5 5.8 12.9 0.9 8.96 7.1 13.3 2.1 2.3 7 6.5 10.2 0.3 4.2 8 9.0 2.9 −0.6 4.5 9 10.0 2.8 −0.86.4 Early ten 6.6 13.3 −0.8 56.0 days of the month 10 3.1 6.2 0.4 7.0 114.3 7.2 1.9 6.4 12 4.0 7.5 −0.6 5.5 13 3.8 8.8 −2.3 8.1 14 5.1 10.4 −1.26.7 15 3.5 6.5 −1.3 2.2 16 3.2 7.8 −1.7 3.0 17 4.4 9.3 0.9 7.8 18 5.512.4 −0.4 6.2 19 8.8 12.6 3.9 1.2 Mid ten 4.6 12.6 −2.3 54.1 days of themonth 20 6.4 12.7 2.1 7.2 21 3.6 8.1 −1.2 6.7 22 3.7 10.5 −1.9 7.6 237.2 13.3 1.2 4.1 24 8.7 13.2 4.6 4.2 25 8.1 12.6 3.1 2.7 26 7.5 11.9 1.08.7 27 5.8 10.7 0.0 2.2 28 6.3 11.4 0.4 7.9 29 3.2 9.0 −0.9 4.9 Late ten6.1 13.3 −1.9 56.2 days of the month 1999 1 30 3.3 9.0 −2.0 9.1 31 5.310.4 −0.6 8.1 2 1 7.6 12.9 3.7 1.3 2 5.7 9.2 1.3 5.1 3 −0.3 1.7 −2.5 7.24 0.1 4.6 −5.2 2.9 5 3.4 6.9 0.5 6.7 6 4.0 9.5 −0.9 8.5 7 5.2 13.0 −0.99.5 8 6.0 14.0 0.5 7.8 Early ten 4.0 14.0 −5.2 66.2 days of the month 96.4 13.8 0.3 8.5 10 7.9 12.8 1.8 5.6 11 6.8 8.3 3.6 2.5 12 4.4 6.3 1.23.6 13 1.9 5.7 −1.9 9.2 14 2.6 9.8 −2.6 8.3 15 4.2 10.4 −0.4 7.2 16 5.712.5 0.0 9.3 17 7.9 15.9 0.8 8.9 18 8.4 10.6 5.3 0.0 Mid ten 5.6 15.9−2.6 63.1 days of the month 19 4.6 7.2 0.4 5.3 20 2.3 4.3 −0.6 3.0 212.7 5.4 −1.9 7.1 22 3.8 8.7 −1.8 1.7 23 6.5 11.2 1.5 5.4 24 7.8 9.6 5.30.0 25 9.6 13.3 5.2 7.3 26 9.0 12.1 3.5 0.0 27 8.4 11.4 2.2 7.3 28 4.810.4 −0.2 9.2 Late ten 6.0 13.3 −1.9 46.3 days of the month 3 1 7.0 12.01.6 5.2 2 9.3 16.4 2.6 5.4 3 9.8 17.4 4.0 10.2 4 8.9 14.6 3.3 1.1 5 14.519.6 10.7 1.5 6 11.0 16.4 5.8 10.0 7 9.3 11.6 7.6 0.0 8 9.4 13.5 7.1 6.79 7.1 8.7 3.8 0.0 10 7.2 10.9 2.9 3.7 Early ten 9.4 19.6 1.6 43.8 daysof the month

Considering the different temperature fluctuation patterns in differentyears, winter was defined as conditions where the mean temperature of aperiod of ten days is around 6° C. or below and the lowest temperatureof this period is −1° C. or below. Since the lower temperature limitcould not be evaluated in the field experiment, a hardening test wasconducted as in Example 3 and FIG. 12, and the lower temperature limitwas found to be −15° C.

“Maintaining green leaves” means maintaining a green to yellow-greencolor without turning brown or dried grass colored or undergoing changeto purplish red, dark green-purple or blackish-purple color even in thefew remaining living leaves during winter, as occurs with conventionalManilagrass, and clearly maintaining a condition recognized as “green”across the entire covered region.

According to the invention, “throughout the year” means that thevarieties of the invention maintain green to yellow-green stems, leavesand spikes throughout the year in periods and regions in whichconventional Manilagrass contains anthocyanins in the stems, leaves orspikes depending on the season and undergoes coloration to purplish red,dark green-purple or blackish-purple, and “containing substantially noanthocyanins” means not only that no purplish red, dark green-purple orblackish-purple coloration is visually found on the ground areas, butalso that anthocyanins are absent (undetectable) or present in onlytrace amounts based on analysis results for the season and plant partsin which conventional Manilagrass undergoes coloration due toanthocyanins, as explained in the examples given below.

According to the invention, the “immature internodes of the front partof the main stolon” are the section in which the internodes, at thefront part of the main stolon including the shoot apex, is stillundergoing vegetative growth.

A “parental strain” according to the invention means a strain used asthe starting material for crossbreeding, mutation, cell fusion or geneintroduction.

As mentioned above, turfgrass has been subjected to seeding methods forthe purpose of increasing green color in winter, and has been used forattempted crossbreeding to shorten the dormant period. However, theresults obtained by such methods have not satisfied the market need.

All conventional Manilagrass, to a widely varying degree depending onthe cultivating region and variety line, experiences withered leaf andbrowning during winter with reduction in chlorophyll in the barelyliving sections, while anthocyanins also accumulate in large amounts,such that blackish-purple coloration occurs. Thus, in order to improvethe greenness in winter it is necessary to increase the living leaves orinhibit decomposition of chlorophyll while inhibiting production ofanthocyanins, while it is also necessary to minimize freezing of livingleaves by frost and prevent any notable chlorosis (decomposition ofchlorophyll); high wear resistance is also preferred for low temperaturegrowth stagnation periods, but no Manilagrass obtained by conventionalmethods has yet exhibited the properties that are the object of thepresent invention. According to the invention, the line having genemutation inducing novel genotypes, characterized by maintaining greenleaves during winter, preventing accumulation of anthocyanin, and havingproperties described in claims 2-3, which are not found in the parentalstrain and any other conventional Manilagrass by genetically mutatingmanipulation on the parental strain, was successfully created.

The Manilagrass of the invention thus contains well-defined purposiveconversion induced in the parental strain line, by a series ofindependent technical systems including a combination of mutationmethods such as cellular mutation, ultraviolet irradiation, X-rayirradiation or the like, formation and use of a kind of shoot primordiaand selection of transmutated cells as a process for creating atransmutated variety with a direction toward the prescribed goal. Theinvention has created completely new genotypes that retain the featuresof Manilagrass such as a narrow blade width and resistance to frozenwithered leaf due to frost, while also: (1) retaining green leaves atlow temperature even though it is Manilagrass, (2) containingsubstantially no anthocyanins, (3) having a short internode length, and(4) having high lateral stolon growth; this has been achieved withoutrelying on conventional methods such as natural hybridization,artificial crossbreeding, cell fusion, gene introduction or selection ofwild varieties. These genotypes are fixed and are not genotypes obtainedby transfer or introduction from other varieties but rather arecompletely new Manilagrass genotypes. The scope of the inventiontherefore encompasses Eragrostoideae plant with the features of theinvention, obtained by adding further mutation using the variety of theinvention, by introducing other genes therein, or by using conventionalmethods such as crossbreeding for introduction of the new genotypes ofthe variety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the changes in anthocyanins in the stolons of Tottori Z.matrella according to HPLC, for Nov. 10, 1997, Nov. 25, 1997, Dec. 23,1997, Jan. 22, 1998 and Feb. 26, 1998 in order from top to bottom.

FIG. 2 shows HPLC analysis of anthocyanin hydrolysates in Tottori Z.matrella at top, and analysis of a standard substance under the sameconditions at bottom.

FIG. 3 shows changes in the anthocyanin composition of leaf blades ofTottori Z. matrella for Nov. 25, 1997, Dec. 23, 1997, Jan. 22, 1998 andFeb. 26, 1998 in order from top to bottom.

FIG. 4 shows changes in the anthocyanin composition of leaf blades ofTK-XG1 for Nov. 25, 1997, Dec. 23, 1997, Jan. 22, 1998 and Feb. 26, 1998in order from top to bottom.

FIG. 5 shows changes in the anthocyanin composition of leaf blades ofTK-XG1 for Nov. 25, 1997, Dec. 23, 1997, Jan. 22, 1998 and Feb. 26, 1998in order from top to bottom.

FIG. 6 is a gel electrophoresis pattern used to distinguish varieties bythe RAPD method using primer A.

FIG. 7 is a gel electrophoresis pattern used to distinguish varieties bythe RAPD method using primer B.

FIG. 8 is a gel electrophoresis pattern used to distinguish varieties bythe RAPD method using primer C.

FIG. 9 is a gel electrophoresis pattern used to distinguish varieties bythe RAPD method using primer D.

FIG. 10 is a gel electrophoresis pattern for TK-XG1 by the AFLP methodusing a combination of 9 primers.

FIGS. 11a to 11 i are electropherograms used to analyze signals detectedwith analysis software after gel electrophoresis of TK-XG1 by the AFLDmethod using a combination of 9 primers.

FIG. 12 is an electrical conductivity (EC) point line graph for TK-XG1and Tottori Z. matrella treated at a low temperature of −15° C. for 0,1.5, 3, 6 and 9 hours. The data are expressed in terms of the rate ofchange in EC due to low temperature treatment (an increase in EC occursupon cellular destruction by freezing), where 1 is defined as the ECbefore treatment at the low temperature of −15° C.

FIG. 13 is a photograph at a research field in which 5-node stolons areplanted, where FIG. 13a is TK-XG1 and FIG. 13b is Tottori Z. matrella.

FIG. 14 is a partial enlarged photograph of FIG. 13, where FIG. 14a isof FIG. 13a and FIG. 14b is of FIG. 13 b.

FIG. 15 is a line diagram showing extension of stolons, where FIG. 15awas drawn from FIG. 14a and FIG. 15b was drawn from FIG. 14b. In eachdrawing, the shaded region is the total-covering dense region. The highlateral stolon growth in the case of TK-XG1 rapidly formed a denseregion with extension of stolons.

FIG. 16 is a comparative line diagram of stolons, cut off 43.0 cm-longfrom their tips of stolons for 8 representative lines.

FIG. 17 is a photograph showing stolen extensions of TK-XG1 and TottoriZ. matrella (see Table 10).

Shown from top to bottom are the stolons for:

A) TK-XG1

B) Tottori Z. matrella

C) Wintercarpet

D) Winterfield

E) Victoria

F) Miyako

G) Meyer

H) Emerald

DETAILED DESCRIPTION OF THE INVENTION

As explained above, in order to solve the different problems of theprior art, i.e. the problems associated with conventional Manilagrassvarieties and lines, the present invention has succeeded in addingintentional mutations to conventional Manilagrass and thereby providingfixed new genotypes. The production method will now be described.

Production Method

After using alcohol or a chlorine-based sterilizer to sterilizeManilagrass mature seed embryo tissue, immature seed embryo tissue andapical growth point, axillary bud growth point or shoot apex tissue, thecellular tissue is collected and planted in a Murashige-Skoog mediumcontaining a plant growth regulating substance such as2,4-dichlorophenoxyacetic acid or indoleacetic acid, and culturing iscarried out in aseptic light or dark conditions while the medium isstationary or vibrated, to obtain shoot primordium (see JapaneseUnexamined Patent Publication No. 6-106427); the shoot primordium areirradiated with high energy such as ultraviolet rays, soft X-rays orgamma rays or immersed in a mutagenic substance to induce geneticmutation and are then further cultured by replanting in theMurashige-Skoog medium, selection is made based on differences in lowtemperature sensitivity, and then after further replanting inMurashige-Skoog medium containing no plant growth regulating substancethe plant bodies are reproduced and grown to young plants. The youngplants are then grown. Acclimatization proceeds very smoothly.

The shoot primordium form domes with a surface structure, and differfrom calli that are void of surface structure. The shoot primordium alsoform aggregates by continued culturing under conditions that producedthe shoot primordium, and when these are planted in solid mediumcontaining no plant growth regulating substance, they can reproduce toplant bodies.

Immersion treatment in a mutagenic substance involves addition anddissolution of a mutagenic substance in a liquid medium, buffer solutionor the like and immersion of the shoot primordium therein to inducemutation. Mutation-inducing mutagenic substances that may be usedinclude 5-bromo-2′-deoxyuridine, ethylmethane sulfonate (EMS),ethyleneimine (EI), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG),N-nitroso-N′-methylurea (MNH), diethylsulfate, 1,2-epoxybutane,2,3-epoxypropionic aldehyde, 8-azaguanine, 5-bromouracil, acridineorange, ICR-10, acriflavin and the like.

EXAMPLE 1 Production of Shoot Primordium

Stolon apical buds in the shoot apex tissue of Tottori Z. matrella, arepresentative Manilagrass line produced and sold by the Tottori SodProducers Association, were collected and promptly rinsed with a 70%(V/V) ethanol aqueous solution, after which they were sterilized in a 1%(V/V) sodium hypochlorite aqueous solution while slowly shaking for 15minutes and thoroughly rinsed with sterilized water, and then the shootapex tissue was planted into solid medium in a vessel by an asepticprocedure in a clean room. The solid medium was a basic medium obtainedby adding 30 g of sucrose and 7 g of agar to 1000 ml of Murashige-Skoogmedium, and further adding 2 mg of 2,4-dichlorophenoxyacetic acid toadjust the pH to 5.8. Culturing was then carried out at 25° C. indarkness. At about 2 weeks after the start of culturing, callicontaining a shoot primordium were obtained as derivatives. The shootprimordium section was cut off and subcultured by the same method toobtain a clump of pure shoot primordium.

Mutation Treatment by Irradiation

A culturing plate was prepared using 20 ml of Murashige-Skoog mediumcontaining 3% sucrose, 0.8% agar and no plant growth regulatingsubstance in a 90-mm diameter plastic Petri dish. The aforementionedclump of shoot primordium cell with a diameter of 2-3 mm was transferredat about 200 per plate and after covering with a quartz disk lid it wassealed with tape. The procedure was carried out aseptically in a cleanroom. The X-ray irradiation apparatus used was a Model OM-100Rirradiating X-ray generator by Ohmic, KK. The ultraviolet ray sourceused was an HP-30C by Atto, KK. having a wavelength of 254 nm and a beamintensity of 1780 μw/cm². The X-ray irradiation intensity was 60 KVp, 4mA, the distance to the target was 500 mm, and a 0.5 mm Al filter wasused for 25 hours of irradiation. A portion of the apparatus wasmodified, and the ultraviolet source was placed in the X-ray irradiationcabinet and irradiation was carried out simultaneously with theultraviolet beam from a distance of 45 cm from the target. Six plateswere irradiated at a time, and the procedure was repeated 10 times fromOctober 2 to Oct. 20, 1995.

Culturing to Young Plants

The irradiation-treated plates were allowed to stand in darkness at 28°C. for 2 days. Selection was then performed by low temperatureculturing. The shoot primordium were transferred onto Murashige-Skoogplate containing 3% sucrose, 0.8% agar and no plant growth regulatingsubstance that had been prepared in a glass bottle with an innerdiameter of 80 mm and a height of 120 mm, and culturing was carried outfor 3 months with a cycle of 16 hours at 15° C. under 10,000 lux lightand 8 hours at 10° C. in darkness.

This low temperature culturing produced green shoot primordium andpurplish red shoot primordium with accumulated anthocyanins. The greenshoot primordium were selected out and transferred onto agar medium ofthe same composition prepared in a wide-rim bottle with an innerdiameter of 80 mm and a height of 120 mm and cultured at 28° C. under4,500-5,000 lux to produce shoots with roots for raising of youngplants.

A total of 6,017 green shoot primordium were found among the 12,800irradiated bodies, and approximately 300 plant were reproduced fromthese up to March of 1996.

Selection of Target Individuals

Each of the young plants obtained by the procedure described above wastransplanted into a #3 pot containing a culturing soil composition ofsand earth:vermiculite:peat moss (Lamex Co.) at 2:1:1, and was allowedto grow outdoors. The soil used contained no fertilizer, but as an addedfertilizer there was provided each week a liquid fertilizer containing 5g nitrogen/10 g phosphoric acid/5 g potassium per 100 g (Householdgardening fertilizer: Hyponex 5-10-10, Hyponex Japan), diluted1000-fold, at 50 ml per pot. In addition, a 2% citric acid soluble slowacting fertilizer containing 6 g nitrogen/38 g phosphoric acid/6 gpotassium/18 g magnesium per 100 g (Greenmap II,M: Nihon Godo Hiryo,KK.) was added at 1 g per pot on Sep. 12, 1996. Irrigation was carriedout daily except for rainy days, for a period of 6 months untilattachment to the soil. Acclimatization proceeded very satisfactorilywithout any special steps. No herbicides, antimicrobial agents orpesticides were used. The outdoor cultivation was at the research fieldof Kaisui Chemical Industry Co., Ltd. at 535 Hamakata, Hofu City,Yamaguchi Prefecture, Japan, and the winter green leaf retention, growthproperties and the period of greening in spring were examined.

The stem color was examined by visually determining the presence orabsence of purplish red pigment formation on May 16, Aug. 10 and Dec. 2,1996. On May 16 the stolons were still undeveloped and the color of thevertical stems was recorded. In the following two examinations the colorof the stolons was recorded.

TABLE 2 Changes in stem color of plants reproduced from shoot primordiumafter irradiation and low temperature selection (1996) Stem color May 16August 10 December 2 Green 6 1 1 Purplish red 239 244 244 Withered 62 6262 Total 307 307 307

Six plants exhibited green vertical stems by the first examination, butin the succeeding examinations only one plant failed to exhibit apurplish red color in both the vertical stems and stolons.

The degree of greenness of the leaves in winter was evaluated visuallyon Dec. 2, 1996 and evaluated based on a 5-level scale.

TABLE 3 Selection based on greenness retained in winter Green- ness Dec.2, 1996 Jan. 31, 1997 Feb. 14, 1997 Feb. 14, 1999 5 2 0 0 0 4 9 1 1 1 3234 0 0 0 2 0 14 11 0 1 0 230 233 234 With- 62 62 62 62 ered Total 307307 307 307 5 - Totally green leaves 4 - Some totally green live leafblades exhibiting no purplish red or yellow color, with some witheredleaf blades exhibiting loss of greenness 3 - Some 1-2 mm blade tipsexhibiting purplish red or yellow color 2 - Greenness remaining only atbase of leaf blades 1 - No greenness throughout entire variety

A variety was selected that exhibited no purplish red pigmentation ofstems or leaves even by February 14 during the above outdoor winterselection, and this variety was designated as Line No. 42-289.

Evaluation of Character

Variety No. 42-289 obtained in the manner described above wastransferred to three planters on Oct. 22, 1996, the line name waschanged to TK-XG1, and the plants were raised in a greenhouse andallowed to grow and observed until the next Jun. 30, 1997. FromSeptember 1997, they were transferred to a research field together with7 control varieties and the characteristics of each were evaluated by a2.0 m square section at 1 location in the dense planted section and at 2locations in the sparse planted section. The soil components of thefield were masatsuchi: native weathered soil from granite and sand in amixture of 7:3, with hard sintered diatomite foamed granules (Isolite:Isolite Industries, KK.) at 100 kg/m³, 2% citric acid soluble slowacting fertilizer at 1.5 kg/m³ and a special fermented fertilizer(Mothersoil: Koto Industries, KK.) at 10 kg/m³. In each dense plantedsection, 81 young plants each with 5-node stolons were transplanted at aspacing of 15 cm. In each sparse planted section, one young plant eachwith 5-node stolons was transplanted at the center. The fertilizer wasevenly dispersed once a month for the 10 months from February toNovember, to a yearly dose of 20 g/m² each for nitrogen, phosphoric acidand potassium.

The characteristics of the dense planted sections examined up toFebruary, 1999 are shown in Table 4. As concerns retention of greenleaves during the low temperature period, TK-XG1 retained green leavesduring the period from May, 1996 to November, 1999, while the Tottori Z.matrella lost its green appearance from mid November as it exhibitedanthocyanin colored leaves due to anthocyanin coloration and witheringof almost all the leaves by late December, and showing dark greencoloration or blackish-purple coloration even in those few living leavesthat remained. The Meyer, Emerald or common Japanese lawngrass (Zoysiajaponica) used for comparison all exhibited purplish coloration due toanthocyanins from mid December, with all of the leaves reaching awithered condition by the end of December. The major object of thepresent invention, which is the feature of producing no anthocyanins,has been visually observed and was substantiated by the analysisdescribed below. The other features were also confirmed as stablecharacteristics.

TABLE 4 Comparison of features of TK-XG1 and Tottori Z. matrella(parental strain, or TK-XG1 starting material) Zoysia matrella Zoysiamatrella (TK-XG1) (Tottori Z. matrella) Anthocyanins Stolons Trace17−min and Anthocyanins detected 20−min HPLC peaks throughout the year;purplish red color Leaves No detectable Detection of same HPLC peakanthocyanins as stolons from autumn through winter Florets Noanthocyanins Dark purple color Anthers No anthocyanins Purplish redcolor Stolon extension (after transplanting 5 nodes previous autumn)Spring-summer Shorter internodes and — more notable lateral stolongrowth than Tottori Z. matrella Early winter Extension of stolonsExtension of stolons from end of Nov. from end of Oct. to to early Dec.early Nov. Winter (greenhouse) New stolons produced No extension ofstolons Stolon density¹⁾ 125.7 82.3 Stolon thickness²⁾ 1.1 mm 1.3 mmSpikes Spike production Spikes produced in Spikes produced all at periodwinter, beginning once in Dec. later than Tottori Z. matrella Bladelength³⁾ 2.3 cm 2.4 cm Blade width⁴⁾ 2.1 mm 2.3 mm ¹⁾Average value at 3locations for the number of stolons crossing a 1.5 m string stretchedparallel to the east-west side of the dense planted section ²⁾Averagevalue of 10 stolons for the diameter at a section between nodes 4 and 5from the tip of the stolons ³⁾Average value of 10 leaves for the bladelength in early summer. ⁴⁾Average value of 10 leaves for the maximumblade width in early summer.

The Tottori Z. matrella had purplish red ears while the TK-XG1 formedyellow-green ears with no purplish red color.

The temperatures at the research field of Kaisui Chemical Industry Co.,Ltd. during the examination period are listed in Table 5.

TABLE 5 Year Mean Highest Lowest or tempera- tempera- tempera- MeanHighest Lowest Mean Highest Lowest Month ture ture ture Monthtemperature temperature temperature Month temperature temperaturetemperature 1996 (° C.) (° C.) (° C.) 1997 (° C.) (° C.) (° C.) 1998 (°C.) (° C.) (° C.) Jan 4.0 13.5 −4.0 Jan 3.7 15.5 −3.5 Jan 4.8 12.0 −4.0Feb 3.0 18.5 −6.0 Feb 5.0 15.3 −3.8 Feb 7.3 19.0 −2.0 Mar 7.2 17.5 −3.5Mar 9.4 18.2 0.0 Mar 8.9 22.5 −1.5 Apr 10.3 26.0 −1.0 Apr 13.3 22.0 0.5Apr 15.9 19.2 13.1 May 17.5 28.5 3.5 May 18.5 27.0 8.0 May 19.7 23.016.0 Jun 21.8 29.0 14.5 Jun 22.1 30.0 14.5 Jun 21.7 24.6 19.4 Jul 26.033.0 17.5 Jul 25.3 32.0 18.5 Jul 27.5 29.7 23.9 Aug 27.3 33.0 20.0 Aug26.8 32.0 20.0 Aug 27.9 31.4 24.9 Sep 22.8 30.0 14.0 Sep 22.9 33.0 12.5Sep 24.4 28.7 21.7 Oct 17.2 26.5 4.5 Oct 16.5 25.0 4.5 Oct 19.5 22.916.3 Nov 12.4 23.0 2.0 Nov 13.2 21.5 1.5 Dec 6.1 15.0 −1.5 Dec 7.6 16.50.0

Confirmation of Anthocyanins by High Performance Liquid Chromatography(HPLC)

The leaf blades and stolons of Tottori Z. matrella and TK-XG1 werecollected separately, and the node sections of the stolons were cut offto leave only the internodes which were then used as samples foranthocyanin analysis. The weight was precisely measured and sea sand wasadded, and after grinding, 30 ml of 0.1% methanol hydrochloride wasadded as an extraction solvent and the mixture was allowed to stand fora day and a night. The extract was filtered with No.2 filter paper andthe extraction solvent was added to 50 ml. For measurement by HPLC (HighPerformance Liquid Chromatography), the filtered substance from amembrane filter (0.45 μm) was used. The elution solvent used was 4%aqueous phosphoric acid (solution A) and acetonitrile (solution B). Thecolumn was equilibrated with 90% solution A and 10% solution B, 10 μl ofsample was added simultaneously with linear increase in the proportionof solution B to reach 70% solution A and 30% solution B after 40minutes. The flow rate of the solution was 1 ml/min, the columntemperature was 40° C., and the column used was a COSMOSil, ODS-C18 byNAKARAI. The detection was performed by measurement of the absorbance at530 nm.

In order to determine the molecular type of the pigment skeleton,hydrochloric acid was added to the extract to create acidity, heatingwas performed at 100° C. for 60 minutes for hydrolysis to remove thesugar chains, and analysis was carried out by HPLC. The elution solventwas at a fixed concentration of 83% solution A and 17% solution B.

When an extract from stolons of Tottori Z. matrella was analyzed by HPLCon Nov. 10, 1997, there were 2 main peaks at 16.930 min. and 20.750 min.(FIG. 1-971110).

When the sample was hydrolyzed and analyzed by HPLC to identify theaglycons of the anthocyanin molecules, a single main peak appeared atthe position of 6.950 min., and a small peak appeared at 13.217 min.(FIG. 2).

Based on the retention time of the standard substance, the main peak wasbelieved to be cyanidin and the small peak peonidin, and the two mainpeaks of FIG. 1-971110 were both concluded to be anthocyanins withdifferent sugar chains on their cyanidin skeletons.

Tottori Z. matrella transplanted at the research field at 535 Hamakata,Hofu City from November, 1997 to February, 1998 was sampled each monthand analyzed (FIG. 1).

The results showed peaks at 16.930 min. and 20.750 min. for the Novembersample, as mentioned above, but the peak at 16.930 min. graduallydecreased becoming a small peak by the end of December. A major peaktook its place at 28.052 min. by the end of January, and was maintaineduntil the end of February. The other main peak in November at 20.750min. was maintained throughout the measuring period though at varyingheights. It was thus demonstrated that the stolons of Tottori Z.matrella are red throughout the year, but that the molecular compositionis not constant but changes with the change in seasons.

Tottori Z. matrella leaves are green in summer, and almost noanthocyanins are detected even when analysis is made at the end ofNovember (FIG. 3). However, anthocyanin colored leaves exhibit acomposition very similar to the anthocyanin composition of stolonscomposed mainly of anthocyanins at 20.795 min. and 28.027 min, that areseen with stolons in February.

The composition of anthocyanins produced in stolons of Tottori Z.matrella (Zoysia matrella) changes with changes in temperature orsunshine, and while the pigment reaches maximum accumulation in earlywinter, eventually reaching the anthocyanin composition of leaves andstolons in the cold season. The winter anthocyanin composition changesas the anthocyanins in the leaves disappear with warming weather, andthe stolons exhibit a warm season anthocyanin composition. Theanthocyanin composition of Tottori Z. matrella traverses this cyclethroughout the year.

The leaves of the TK-XG1 of the invention produced no anthocyanins whenmeasured from November, 1997 through February, 1998 (FIG. 4). Two tracepeaks were detected in the stolons at 16.908 and 20.792 on November 25,while only trace peaks were detected at 17.225, 20.725 and 28.075 onJanuary 22, and at 20.725 and 27.840 on February 26 (FIG. 5).

Thus, Tottori Z. matrella accumulates the red pigment anthocyanins inthe leaves in winter, whereas the new variety TK-XG1 accumulates noanthocyanins in the leaves during winter. Moreover, anthocyanins areproduced in the stolons in only trace amounts, and may therefore beconsidered to contain substantially no anthocyanins. Outwardly, TK-XG1exhibited no anthocyanin coloring at any part or in any season.

Determination of Anthocyanin Molecular Structures

TK-XG1 contains only trace amounts of molecules at 17 minutes and 20minutes among the anthocyanin molecules typically appearing inManilagrass and, therefore, it possesses, as a major feature, arelatively substantial absence of anthocyanins. In order to elucidatethe molecular structures of the anthocyanins, Tottori Z. matrella wasused to determine the structures of anthocyanins in Manilagrass.

The molecular structures were determined by the method ofco-chromatography, and as a result it was found that the principalanthocyanins in Manilagrass are cyanidin 3-glucoside (Cy3G) at 10 min.,cyanidin 3-malonylglucoside (Cy3MG) at 17 min., cyanidin3-dimalonylglucoside (Cy3DMG) at 20 min. and cyanidin3-polymalonylglucoside (Cy3PMG) at 28 min.

Chlorophyll Concentration in Winter

The changes in chlorophyll concentration in TK-XG1 and the Tottori Z.matrella parental strain were measured between September, 1997 andMarch, 1998 (see Table 6).

TABLE 6 Chlorophyll contents in Tottori Z. matrella and TK-XG1 Zoysiamatrella Zoysia matrella Sampling (Tottori Z. matrella) (TK-XG1) dateChl a Chl b Total Chl a Chl b Total Yr Mon Day (mg/g) (mg/g) (mg/g)(mg/g) (mg/g) (mg/g) 97 09 29 1.11 0.44 1.55 1.48 0.60 2.09 97 10 131.16 0.42 1.57 1.83 0.57 2.40 97 10 29 0.98 0.35 1.37 1.85 0.59 2.43 9712 10 0.91 0.32 1.23 1.32 0.49 1.81 98 01 27 1.06 0.38 1.42 1.25 0.421.68 98 02 12 0.82 0.25 1.07 1.04 0.32 1.37 98 03 24 1.15 0.41 1.56 1.350.44 1.79

The chlorophyll was extracted from the leaf blades with 80% acetone, theabsorbance was measured at 645 nm and 663 nm, and the content wascalculated by the Arnon formula (Arnon, 1949, Plant Physiology, 24,1-15). The Tottori Z. matrella suffered winter withering in winter, witha content of less than 1% in the living leaves. Since only the livingleaves were selected for use, excluding the withered leaves, the TottoriZ. matrella had a higher value than expected for the visual sensationappearance of total winter withering, but it still had a significantlylower chlorophyll content in winter than TK-XG1.

DNA analysis of TK-XG1 by RAPD Method

In order to clearly elucidate the differences between TK-XG1 and othervarieties, analysis was performed by the RAPD (Random AmplifiedPolymorphic DNA) method (see FIGS. 6-10). Nine varieties were used,TK-XG1 and eight controls: Tottori Z. matrella, winterfield (Zoysiamatrella, registered variety in Japan), Wintercarpet (Zoysia matrella,registered variety in Japan), Victoria (also known as Preciousgreen.Zoysia japonica, plant patented in U.S.), Miyako (Zoysia japonica×Zoysiamatrella or Zoysia japonica, registered variety in Japan), Meyer (Zoysiajaponica, registered variety in U.S.), Emerald (Zoysia japonica×Zoysiatenuifolia, registered variety in U.S.) and El Toro (Zoysia japonica,plant patented in U.S.), were cultivated under the same conditions asthe research field at Kaisui Chemical Industry Co., Ltd., and the samevolume of leaves were sampled on the same day and the DNA extracted bythe CTAB (Cetyltrimethylammonium bromide) method. The DNA was used as atemplate for PCR (Polymerase Chain Reaction) using primers with thesequences given below. The reaction conditions were 2 minutes at 94° C.for pre-amplification heat denaturation, 30 seconds at 94° C. fordenaturation for the amplification cycle, 30 seconds at 34° C. forannealing and 2 minutes at 68° C. for extension. This cycle was repeated45 times for reaction, followed by a final extension for 5 minutes at68° C. A 2% agarose gel was used for electrophoresis. The primers forthe photographs shown as data were the four primers with the followingsequences

Primer A: TTCCGTAATCAC (FIG. 6)

Primer B: AGAGGTGTAAAT (FIG. 7)

Primer C: TTGCATAATCGT (FIG. 8)

Primer D: CCTTGGAACTCG (FIG. 9)

TK-XG1, Tottori Z. matrella and the other varieties can be distinguishedby comparison of the electrophoretic images of their PCR amplificationproducts using primers A, B and C, for example (see FIGS. 6, 7, 8). TheTottori Z. matrella produces a band at 1330 bp by amplification withprimer D, and since this is absent with TK-XG1 this can be used todistinguish the two varieties (see FIG. 9).

The base sequences of the primers used for PCR amplification in FIGS. 6to 9 have been given above. Lanes 1) and 11) in FIGS. 6, 7 and 8 aremolecular weight markers at 250 bp spacings, and the 1000 bp bands areindicated by an arrow. The other lanes are 2) TK-XG1, 3) Tottori Z.matrella, 4) Wintercarpet, 5) Winterfield, 6) Victoria, 7) Miyako, 8)Meyer, 9) Emerald and 10) El Toro. Lanes 1) and 4) in FIG. 9 aremolecular weight markers, lane 2) is TK-XG1 and lane 3) is Tottori Z.matrella.

DNA Analysis of TK-XG1 by AFLP Method

In order to clearly elucidate the differences between TK-XG1 and othervarieties, analysis was performed by the AFLP (Amplified Fragment LengthPolymorphism) method. Leaves were collected from TK-XG1 cultivated atthe research field at Kaisui Chemical Industry Co., Ltd., and the DNAwas extracted by the CTAB (Cetyltrimethylammonium bromide) method. Aftercutting the DNA with two different restriction endonucleases EcoRI(6-base recognizing enzyme) and MseI (4-base recognizing enzyme), adouble-stranded adapter was linked to both ends of the DNA fragmentwhich was then subjected to Preselective PCR (Polymerase Chain Reaction:preselective amplification) first and then a preselective primer wasused to add one additional base downstream from the restrictionendonuclease site and only the matching bases of the restrictionendonuclease fragment were selectively amplified. The reactionconditions were one cycle of 2 minutes at 72° C. for pre-amplificationheat denaturation, followed by 20 cycles of 20 seconds at 94° C. fordenaturation for the amplification cycle, 30 seconds at 56° C. forannealing and 2 minutes at 72° C. for extension. A 1.2% agarose gel wasused for electrophoresis to identify the PCR product. A fluorescentdye-labeled selective primer was then used for a second PCR (selectiveamplification) reaction. The reaction conditions are shown in Table 7. Amixture of the second PCR product, loading dye and ROX500 Size Standardwas used for the electrophoresis sample. A DNA sequencer (ABI PRISM377DNA Sequencer) was used for fractionation with 6% polyacrylamide gelelectrophoresis (FIG. 10), after which analysis software (ABI PRISM GeneScan Analysis) was used for analysis of the detected signal on anelectropherogram (FIG. 11a to FIG. 11i). The experiment was repeated 3times.

TABLE 7 Reaction conditions for second PCR by AFLP method 94° C. 2 min.94° C. 20 sec. 66° C. 30 sec. 72° C. 2 min. 1 cycle 94° C. 20 sec. 65°C. 30 sec. 72° C. 2 min. 1 cycle 94° C. 20 sec. 64° C. 30 sec 72° C. 2min. 1 cycle 94° C. 20 sec. 63° C. 30 sec. 72° C. 2 min. 1 cycle 94° C.20 sec. 62° C. 30 sec. 72° C. 2 min. 1 cycle 94° C. 20 sec. 61° C. 30sec. 72° C. 2 min. 1 cycle 94° C. 20 sec. 60° C. 30 sec. 72° C. 2 min. 1cycle 94° C. 20 sec. 59° C. 30 sec. 72° C. 2 min. 1 cycle 94° C. 20 sec.58° C. 30 sec. 72° C. 2 min. 1 cycle 94° C. 20 sec. 57° C. 30 sec. 72°C. 2 min. 1 cycle 94° C. 20 sec. 56° C. 30 sec. 72° C. 2 min. 20 cycles60° C. 30 sec. Stored at 4° C.

Confirming Fixation by Large-scale Culturing with Multiple Shoots

Culturing of the growth points of TK-XG1 in Murashige-Skoog mediumcontaining 0.02 mg/l NAA and 0.2 mg/l BA produces multiple shoots(Japanese Unexamined Patent Publication No. 7-313008). The multipleshoots are aggregates of buds, and culturing of a single multiple shootin a large-sized vessel under the same conditions results in growth toabout 100 multiple shoots within two months. The multiple shoots canproduce shoots with roots to yield about 10,000 young plants. When thesewere transferred to a research field and raised, none of the plantsshowed a transformation to the TK-XG1 character. That is, the characterof each apical bud was stable even when grown to 10,000 plants, and thecharacter was even stable with growth at the field from September, 1997and later, or at least the useful character described above waspreserved with vegetative propagation.

The present inventors therefore designed production of “Manilagrass thatretains green leaves in winter”, and as the method used to realize thisgoal, highly differentiated shoot primordium cultured cells capable ofreproduction through callus formation were prepared and irradiated with,for example, ultraviolet rays or soft X-rays to introduce the necessarymutations, and a special cell selection method was adopted and carriedout whereby the green colored cells were selected, to obtain theoriginally targeted new turfgrass variety.

EXAMPLE 2 Observation of Retained Green Leaves of TK-XG1 in Chugoku areaCold District

A test was conducted to evaluate TK-XG1 and Tottori Z. matrella at theHiroshima Prefectural University Agricultural Research station (562Nanatsukahara, Shobara City, Hiroshima Prefecture, Japan) during theperiod from late November, 1998 to early March, 1999. During thisperiod, the TK-XG1 clearly retained its green leaves, but the Tottori Z.matrella produced anthocyanin colored leaves and withered leaf by midDecember and could not retain its green color (Table 8).

TABLE 8 Observation of retained green color of TK-31 XG1 in Chugoku areacold district (Hiroshima Prefectural University: 562 Nanatsukahara,Shobara City, Hiroshima Prefecture) Date Variety Nov. 24, 1998 Dec. 9,1998 Dec. 23, 1998 Jan. 28, 1999 TK- 5 5 4 4 XG1 Tottori 5 3 2 2-1 Z.matrella 5 - Totally green living leaves 4 - Reduced green color, withsome withered leaves 3 - Partially anthocyanin colored or purplish redleaves 2 - Totally anthocyanin colored or purplish red leaves 1 -Withered leaves

The weather conditions at the research station during the test periodare shown in Table 9.

TABLE 9 Weather conditions from Dec. 11, 1998 to Mar. 10, 1999 inChugoku area cold district (Hiroshima Prefectural University: 562Nanatsukahara, Shobara City, Hiroshima Prefecture) Mean Highest LowestSunshine temperature temperature temperature time Yr.Mon.Day (° C.) (°C.) (° C.) (hr) 1998 12 11 1.9 6.3 −1.6 2.6 12 3.2 9.8 −1.2 8.5 13 2.612.1 −5.1 6.7 14 4.8 11.4 −1.7 4.8 15 6.5 9.7 1.9 5.7 16 5.2 11.7 0.46.7 17 4.6 13.8 −2.1 6.9 18 3.5 12.5 −3.2 7.2 19 4.0 13.6 −2.2 4.9 206.2 11.6 0.2 7.8 Mid ten 4.3 13.8 −5.1 61.8 days of the month 21 2.610.8 −3.2 7.7 22 3.8 13.6 −3.9 7.9 23 4.0 11.8 −2.4 5.3 24 4.5 8.4 1.24.8 25 2.9 9.7 −2.6 8.6 26 4.1 11.3 0.0 5.4 27 2.4 6.5 −2.0 0.7 28 5.011.3 0.4 4.9 29 3.4 10.1 −2.2 3.5 30 3.2 7.7 −0.1 7.3 Late ten 3.6 13.6−3.9 56.1 days of the month 12 31 1.3 5.8 −0.8 3.6 1999 1 1 0.8 3.8 −1.21.4 2 −0.3 2.8 −2.2 1.2 3 1.7 7.8 −1.8 3.1 4 1.6 8.9 −1.9 5.2 5 1.1 11.0−5.0 7.1 6 2.7 9.4 −4.4 5.6 7 1.8 5.7 −3.9 2.9 8 −4.4 −3.1 −5.2 3.9 9−3.9 −1.1 −6.3 4.6 Early ten 0.2 11.0 −6.3 38.6 days of the month 10−1.5 0.9 −4.2 4.1 11 0.1 3.2 −1.3 3.7 12 −0.7 2.5 −2.5 5.4 13 −0.5 4.0−5.9 5.8 14 1.1 7.0 −3.4 7.1 15 −0.4 2.9 −3.2 2.1 16 −0.4 5.0 −5.1 4.417 1.6 7.1 −2.9 8.9 18 1.0 9.6 −4.5 5.4 19 1.7 3.9 −1.2 0.0 Mid ten 0.29.6 −5.9 46.9 days of the month 20 2.4 7.0 0.1 3.6 21 0.6 5.5 −3.6 6.222 0.4 9.4 −7.2 8.6 23 4.0 9.9 −2.2 4.5 24 5.0 10.5 0.0 3.3 25 3.8 10.2−1.2 2.1 26 3.9 8.7 −3.0 8.1 27 1.8 9.0 −5.1 5.6 28 2.1 7.4 −3.6 5.8 29−1.1 2.9 −3.8 4.0 Late ten 2.3 10.5 −7.2 51.8 days of the month 1999 130 −0.3 5.0 −4.0 6.3 31 1.3 9.5 −4.9 8.5 2 1 3.3 8.5 −0.2 1.8 2 −0.2 4.5−2.8 2.7 3 −4.7 −2.3 −7.4 3.7 4 −4.6 2.0 −10.7 7.2 5 −1.8 1.7 −5.1 4.8 6−0.8 5.2 −4.9 7.7 7 0.5 8.4 −6.3 9.1 8 0.9 9.0 −5.7 6.2 Early ten −0.69.5 −10.7 58.0 days of the month 9 2.7 11.5 −3.9 8.1 10 3.4 9.7 −1.7 6.211 1.8 4.7 −0.6 1.2 12 −1.1 1.2 −3.3 6.4 13 −2.5 −0.1 −5.0 6.3 14 −1.54.0 −5.2 6.9 15 −0.7 4.5 −4.9 2.1 16 1.8 11.5 −4.3 7.4 17 4.2 13.5 −4.68.9 18 3.3 5.3 1.5 0.0 Mid ten 1.1 13.5 −5.2 53.5 days of the month 190.2 2.2 −2.6 3.1 20 −1.4 0.7 −3.0 6.4 21 −2.0 1.3 −4.1 7.7 22 −0.9 5.6−6.7 8.3 23 2.5 8.4 −2.5 8.3 24 2.4 4.5 −0.1 0.0 25 5.6 12.5 0.4 8.1 263.3 8.1 −2.7 0.3 27 4.0 7.5 −1.0 5.4 28 2.2 9.2 −2.7 8.5 Late ten 1.612.5 −6.7 56.1 days of the month 3 1 3.5 11.0 −2.7 8.8 2 5.2 13.1 −1.66.7 3 6.5 15.5 −1.7 10.1 4 5.8 15.0 −2.3 5.7 5 10.1 16.5 5.7 1.7 6 8.415.2 2.6 9.9 7 5.2 6.4 3.8 0.0 8 5.5 11.4 2.1 7.2 9 3.2 4.2 1.0 0.0 105.3 12.3 0.8 7.7 Early ten 5.9 16.5 −2.7 57.8 days of the month

EXAMPLE 3 Hardening Test

After one month of raising TK-XG1 and Tottori Z. matrella established ina wagnel pot in an artificial weather apparatus (LH-200RDZ, NipponMedical & Chemical Instruments Co, Ltd.) at 4±2° C. under 50,000 lux, alow temperature resistance test was conducted in a freezer (MDF-293AT,SANYO). Approximately 50 mg of live leaves were collected, weighed andwrapped in aluminum foil, and after freezing in freezers set to −15° C.and −10° C. for periods of 0, 1.5, 3, 6 and 9 hours, each sample wastaken out, immersed in 20 ml of distilled water and allowed to stand for24 hours in a refrigerator (5° C.), and an electric conductivity meter(Model SC82 Personal SC Meter, YOKOGAWA) was used to measure theelactric conductivity (EC); the EC values for the Tottori Z. matrellaand TK-XG1 prior to the start of low temperature treatment were eachdefined as 1.0, and the changes in time were observed to evaluate thedegree of freezind-induced cell disruption. Almost no cell disruptionoccurred in either the TK-XG1 or Tottori Z. matrella when treated at−10° C., but when treated at −15° C. both exhibited an increase in ECdue to cell disruption (FIG. 12). However, TK-XG1 showed a gentler slopefor the EC up to 3 hours of low temperature treatment compared to theTottori Z. matrella, suggesting that it was more resistant tofreezing-induced cell disruption.

EXAMPLE 4 Growth Test with TK-XG1

Soil comprising a mixture of masatsuchi and sand in a ratio of 7:3 witha mixture of 100 kg/m³ of hard foaming sintered diatomite granules, 1.5kg/m³ of citrate soluble slow acting fertilizer and 10 kg/m³ of aspecial fermented fertilizer (research field, Kaisui Chemical IndustryCo., Ltd. at 535 Hamakata, Hofu City, Yamaguchi Prefecture) was used,and 5-node stolons were transplanted at the center of a 2.0 m squaresection on Sep. 17, 1997 and supplied with 12.5 g/m² of a chemicalfertilizer containing nitrogen:phosphoric acid:potassium at 10:10:10 wt% (Starmine: Nitto FC Co., Ltd.) at a frequency of once a month topromote growth. The full photographs on Oct. 19, 1998 are shown in FIG.13a (TK-XG1) and FIG. 13b (Tottori Z. matrella). Representativepartially enlarged photographs are shown in FIG. 14a (TK-XG1) and FIG.15b (Tottori Z. matrella), and line diagrams showing the extension ofstolons therefrom are shown in FIG. 15a (TK-XG1) and FIG. 15b (TottoriZ. matrella). Also, FIG. 16 shows a comparative line diagram of 43.0cm-long representative stolon tips for 8 lines grown in the same manner.

Turfgrass internode length and stolon length are determined by thegrowing conditions. For example, the internode length of stolons thatfreely extend from the soil surface tends to be shortened, and thegrowth of lateral stolons is often suppressed. Growth is also suppressedwhen the stolons overlap or are inhibited by some physical obstacle.Spindly growth usually results from shading from sunlight. For anaccurate comparison, therefore, it is necessary to collect and comparestolons extending under the same growth conditions, attached to the soilsurface and free from obstacles. The front part of the stolons weretaken to a length equal to 20 nodes (43.0 cm) of TK-XG1 including theshoot apex. Tables 10 and 11 show the internode distances and stolonextension as actually measured based on FIG. 16. In this example, asshown in FIG. 16, the still extending immature internodes were withinabout 10 cm, and this range was excluded from the measurement.

TK-XG1 was compared with its parental strain (starting material formutagenic manipulation), the Tottori Z. matrella, and found to haveroughly the same extension overall (see FIGS. 13a, 13 b), but itsshorter internode distance and clearly greater lateral stolon growthresulted in a notably larger area of coverage (dense planting section)(see FIGS. 14-a, -b and FIGS. 15a, 15 b). A shorter internode distanceand greater lateral stolon growth increases the covering speed to form adenser ground cover, while the strong creeping property of TK-XG1(property of attaching to soil and extending) increases the overlap ofstolons in the ground and enhances the soil surface protection; therecovery after injury to the grass layer surface by spike injury and thelike is also more rapid, a turf layer with excellent elasticity can beformed, and thus in practical terms it is possible to provide turfgrassof much higher value than conventional Manilagrass (Zoysia matrella) orother Eragrostoideae turfgrass.

For data processing, one maximum and minimum value of the measuredinternode lengths for each variety were discarded and only the remainingvalues were used, in order to eliminate the abnormal values. Consideringthe different sensitivities of the different plant varieties dependingon the environmental conditions, soil form and fertilization method, theratio of internode length of TK-XG1 was in the range of 0.938-0.651 withrespect to 1.0 for Tottori Z. matrella and Meyer, and was significantlydwarfed. Both Tottori Z. matrella and Meyer were used as comparativecontrols because they are representative turfgrass varieties of thesubfamily Eragrostoideae and the internode measurement data wasplenteous for this example, so that they could serve as accuratestandards for data processing. Incidentally, since Eragrostoideaeturfgrass generally undergoes varying internode growth depending on thegrowing conditions, it is preferred for comparative evaluation to becarried out according to this example; however, it is at least necessaryto compare the stolons under conditions in which they are attached tothe soil surface and extend without obstacles. It is also necessary toselect stolons with continued growth without injury to the shoot apex.The internode near the tip of the main stolon lengthens with growth andextension of the main stolon, and internode extension ceases withmaturity. It is therefore necessary to make the comparison of the areanear the tip end excluding the immature tip. For this example, as shownin FIG. 16, the still extending immature internodes were about 10 cm,and these sections were excluded from measurement of the internodelengths. In practice it is preferred for the lateral stolons to followup near to the tip end of the main stolon to create a dense vegetativeregion. As shown in Table 4, the blade width was narrower and the bladelength was slightly shorter compared to its parental strain Tottori Z.matrella.

The ratio of the main stolon growth to the total lateral stolon growthwas in the range of 1.2-1.4 times that of the Tottori Z. matrella and1.4-1.8 times that of the Meyer.

TABLE 10 Internode length of vegetative stolons attached to soilsurface, excluding part of a stolon, cut off 10 cm long from its tipInternode length (mm) from section excluding 100 mm tip (330 mm) 1 2 3 45 6 7 8 9 10 11 TK-XG1 26.4 25.9 28.4 25.5 24.8 (24.6) 26.3 24.9 27.3(29.8) 28.8 Tottori (17.5) 32.6 38.1 (42.5) 36.6 34.9 36.9 30.7 — — — Z.matrella (1) Wintercarpet (2) 48.7 49.1 51.7 (52.2) 44.2 (40.6) — — — —— Winterfield (3) 41.1 35.7 38.0 39.6 36.3 (34.4) 40.5 (41.1) — — —Victoria (4) 36.5 (40.1) 40.1 40.1 37.2 (35.0) 39.9 — — — — Miyako (5)74.6 (69.3) 76.5 (79.9) — — — — — — — Meyer (6) 34.7 32.5 (39.5) 38.938.9 34.8 35.1 (27.3) 32.3 Emerald (7) (40.9) 41.9 43.4 44.2 (46.5) 45.141.9 — — — — Average of Ratio of Ratio of values maximum for minimum forexcluding TK-XG1 and TK-XG1 and abnormal minimum of maximum of valueseach control each control Av. [R] variety variety TK-XG1 26.5 [4.0]Tottori 35.0 0.938 0.651 Z. matrella (1) [7.4] Wintercarpet (2) 48.4 — —[7.5] Winterfield (3) 38.5 — — [5.4] Victoria (4) 38.8 — — [3.6] Miyako(5) 75.6 — — [1.9] Meyer (6) 35.3 0.892 0.638 [6.6] Emerald (7) 43.3 — —[3.2] (1) A Manilagrass (Zoysia matrella) variety produced by theTottori Sod Producers Association: starting material for TK-XG1 (2)Acinic Manilagrass developed by Sumitomo Metal Industries, KK. (3) Sameas above (4) Improved variety bred by Dr. V. Giveault et al., obtainedby crossbreeding Japanese turfgrass with El Toro (an improved variety ofZoysia japonica) as the parent. (5) Hybrid variety of Zoysia japonica ×Manilagrass, bred by Japan Turf, KK. (6) Line selected from Zoysiajaponica seeds, bred by cooperation between the U.S. Dept. ofAgriculture test grounds and the Golf League Green Section (7) Lineselected from hybrid variety of Zoysia japonica and Zoysia tenuifolia,jointly bred by the U.S. Dept. of Agriculture and the All-American GolfAssociation (8) The maximum and minimum value ratios were alsocalculated after excluding the maximum and minimum values (in brackets)for the internode lengths for each variety, in order to avoidinordinately large ranges due to abnormal values. (9) The underlinedmeasured values are the maximum and minimum values of the internodelengths after exclusion of the abnormal values (in brackets).

TABLE 11 Ratios of lengths of main stolons which are cut off 43 cm fromtheir tips to lengths of total lateral stolons of growing stolonsattached to the soil surface Total Lateral lateral stolon/main Totalstolon stolon ratio for lateral length/main TK-XG1 with stolon stolonrespect to 1.0 length¹⁾ length for control (cm) 43 cm variety²⁾ TK-XG1{circle around (1)} 111.6 2.60 — {circle around (2)} 118.4 2.75 —{circle around (3)} 101.9 2.37 — Tottori {circle around (1)} 84.1 1.961.33 Z. matrella {circle around (2)} 89.1 2.07 1.33 {circle around (3)}81.3 1.89 1.25 Wintercarpet {circle around (1)} 35.4 0.82 — {circlearound (2)} 34.2 0.80 — {circle around (3)} 36.7 0.85 — Winterfield{circle around (1)} 12.0 0.28 — {circle around (2)} 12.5 0.29 — {circlearound (3)} 11.3 0.26 — Victoria {circle around (1)} 15.1 0.35 — {circlearound (2)} 15.7 0.37 — {circle around (3)} 14.2 0.33 — Miyako {circlearound (1)} 8.3 0.19 — {circle around (2)} 8.5 0.20 — {circle around(3)} 8.1 0.19 — Meyer {circle around (1)} 63.5 1.48 1.76 {circle around(2)} 65.7 1.53 1.80 {circle around (3)} 71.4 1.66 1.43 Emerald {circlearound (1)} 33.6 0.78 — {circle around (2)} 34.1 0.79 — {circle around(3)} 39.8 0.93 — ¹⁾{circle around (1)}, {circle around (2)} and {circlearound (3)} are the series numbers for each experiment group.²⁾Calculated for each experiment group in the following manner: forexample, for experiment group {circle around (1)}, 2.60/1.96 = 1.33.

EXAMPLE 5 Production of Hybrid Variety Inheriting the Character ofTK-XG1 of the Invention

In order to confirm that the new character of the TK-XG1 of theinvention can be inherited and transferred by crossbreeding withEragrostoideae plant, it was crossbred with Miyako (hybrid variety ofZoysia japonica, by Japan Turf, KK.) in an attempt to produce aderivative variety.

As a result, the Miyako that was crossbred with the TK-XG1 exhibited adark purplish red color in the stolons due to anthocyanins under thesame conditions, whereas the newly produced variety had yellow-greenstolons with substantially no accumulation of anthocyanins, as one ofthe unique characteristics of TK-XG1. The production process and someproperties of the new artificially crossbred variety produced therebywill be described below.

Crossbreeding was carried out with Miyako as the female and TK-XG1 asthe male. The crossbreeding method was according to “AgriculturalExperimental Manual of Plant Production” (Hinata, Y. and Hashibia, T.ed., 1995, Softscience Publications, pp.263-268).

After crossbreeding, the product was raised and matured in a greenhouseand seeds were collected after 40 days. After indoor storage for 3months to improve the germinating rate of the collected seeds, thefertile seeds and non-fertile seeds were separated by the alcoholselection method. A total of 98 settling seeds were selected for use andthe germinating rate was increased by treatment with 0.5% NaOH (seeHirayoshi, I., Matsumura, M., Iwata, E., “Dissemination and Growth ofUseful Wild Grasses”, Gifu University, Agricultural Dept. LaboratoryReport, No.28, 1969, pp.239-251).

The 98 seeds treated with 0.5% NaOH were placed on water-absorbed filterpaper stretched over a Petri dish, and a germination experiment wascommenced under conditions of 35° C., 8,500 lux in an artificial weatherapparatus.

A total of 23 seeds germinated among the 98 seeds, and on the fifth dayafter germination the young buds were transferred to plastic potscontaining autoclave-sterilized rice cultivating soil (Kumiai UbeSpecial Soil #2 by Ube Industries, Ltd., containing 0.2 g each ofnitrogen and phosphoric acid and 0.33 g of potassium per kg).

The eighteen seedlings that were raised from the 23 germinated seedswere transferred to growing pots. The cultivating soil composition ofthe transfer pot soil was a mixture of sea sand:hard forming sintereddiatomite granules:peat moss:woody compost (bark) at 6:2:1:1, alsocontaining 2% citric acid soluble slow acting fertilizer at aconcentration of 1.5 kg/m³, and the plants were raised with watersprinkling in a greenhouse at 28° C.±3° C. The 18 young plants were setin the same section and 125 fruiting seeds were collected, stored,selected and germinated by the same method to obtain 19 young plants. Ofthese, one variety was obtained having stolons exhibiting no purplishred color and containing no anthocyanins. The blade width, internodelength and stolon thickness of the resulting young plant of this varietywere measured on Nov. 22, 1999. Table 14 shows the data for the varietythat exhibited no purplish red color in the stolons.

It is known that crossbred varieties can be produced, even in the caseof distant relative crossbreeding, so long as the plants have a matchingbase number of chromosomes (See, for example, “Turfgrass and ItsVarieties”, pp.126-130 cited above.), and this example demonstrates thepossibility of developing crossbred varieties that inherit the inventedgenetic character of TK-XG1.

TABLE 14 ♀Miyako × ♂TK-XG1: Growth conditions of variety F₂ (observedNov. 22, 1999) F₂ Variety exhibiting no purplish red ♀Miyako ♂TK-XG1color Purplish red color present absent absent in stolons Blade width(mm) 3.6 2.0 2.5

It is not clear whether the various characteristics of the new varietyof the invention each appeared independently or whether a mutation in asingle gene expresses these various phenotypes, but in generalconsideration of mutation methods it may be more naturally assumed thatthe completely new multiple Manilagrass characteristics exhibited by thevariety of the invention are independent characteristics due tomutations at multiple loci. Each new characteristic is absent from theparental strain and each is independently novel, practical and useful.By using these individual genotypes in variety-producing methods such ascrossbreeding, it is possible to produce derivative varieties with newpractical uses, utilizing the four aforementioned genotypes of theinvention.

Comments for Cultivation

The TK-XG1 of the invention can be cultivated at production field by thesame methods as Tottori Z. matrella. However, because of its highergrowth rate compared to Tottori Z. matrella, it is preferably given aslight excess of fertilizer while observing the condition of growth.Growth by multiple shoots is also possible. For practical use, it can beeasily cultivated at the same production field by the same methods asTottori Z. matrella. Cultivation management can also reinforce certaincharacteristics required by the purpose for which the TK-XG1 of theinvention is to be used.

I claim:
 1. The TK-XG1 variety of Manilagrass deposited as FERM BP-7837characterized by retaining its green leaves under a condition where themean temperature of a period of ten days is 6° C. or below and thelowest temperature in this period is −1° C. or below, but not less than−15° C., and by containing substantially no anthocyanins throughout theyear, and by that the length of internode of the main stolon except theimmature internode of the front part of the main stolon, which extendswhen attached to the soil surface under obstacle-free growth conditions,is about 0.9—about 0.6, where 1.0 is defined as the length forconventional Tottori Z. matrella, and also by that the ratio of the mainstolon length to the total lateral stolon length, measuring the totallength of lateral stolons developing from the main stolon based on amain stolon length corresponding to at least 20 nodes from the tip ofthe main stolon of the turfgrass of the invention, in stolons extendingunder obstacle-free growth conditions when attached to the soil surface,is at least 1.2 times compared to conventional Tottori Z. matrella.